One of the major immune responses that protects a host from disease, especially from intracellular infection, is the generation of cytotoxic T lymphocytes (CTLs). CTLs kill host cells that are infected and thereby eliminate the production and/or reservoir of the pathogen. CTLs may also control secondary pathogenic effects induced by infectious organisms, for example, the growth of transformed cells. There is abundant evidence that CTLs are critical components in the defense of the host against several viral pathogens, including influenza, POX and Herpes (Blanden, Transplant Rev., 19:56 (1975); Yap et al., Nature, 273:238 (1978). Furthermore, CTLs can provide immunity in vivo to retrovirally-induced diseases (Earl et al., Science, 234:728 (1986)). There is increasing evidence that CTLs may play a role in protection from human immunodeficiency virus (HIV). For example, CTLs from infected humans and apes can lyse infected cells and inhibit virus production in vitro.
One of the most efficacious and cost effective therapies for the prevention of infectious diseases is the stimulation of specific immune response through vaccination. The need for an effective HIV vaccine is tragically apparent.
Historically, vaccines have been prepared by killing or attenuating a pathogen, such as a virus or bacterium, and then injecting the resulting particles into a patient or host animal. Vaccines have also been prepared by using only a portion of the pathogenic organism, such as a predominantly important protein subunit from a bacterium or virus. While non-living virus particles and subunit vaccines can prime for a class-II restricted antibody response, such non-living vaccines typically do not prime for cytotoxic T lymphocyte immunity.
It is now well established that the tight segregation of the MHC class I pathway of antigen presentation accounts for the failure of most protein-based vaccines to prime CTL responses and is a major obstacle to using such vaccines. Specifically, from the published literature it is known that most antigens in the extracellular fluid are taken up by specialized antigen presenting cells (APCs), processed in an endosomal compartment, and subsequently displayed in association with class II MHC molecules, which elicits an antibody response. In contrast, most exogenous antigens are not presented in association with class I MHC molecules, which is necessary for a class I-restricted CTL response. However, exogenous antigens are presented in association with class I molecules if they are introduced, via experimental manipulations, into the cytoplasm ("cytosol") of cells. It is thought that the antigenic peptides that arise from processing in the cytosol are transported to the endoplasmic reticulum where they associate with class I MHC molecules. The failure of exogenous proteins to be presented in association with class I molecules reflects the inability of these proteins and their degraded endosomal products, to communicate with the appropriate cytosolic compartment, under physiological conditions. As a consequence of this segregation between MHC-class I and class II antigen-presentation pathways, CTLs are selectively targeted to pathologically-affected cells (ie. cells synthesizing abnormal proteins). Uninfected, healthy cells are not at risk of elimination when they encounter antigens in the extracellular fluids.
Despite the generally recognized inability of most antigens to prime CTL response, there have been reports in literature of inducing MHC class I-restricted CTLs with non-replicating antigen in vivo. For example, Zhou et al. have shown that allogeneic splenocytes, MHC-free red blood cells, and synthetic lipid vesicles (liposomes) loaded with chicken ovalbumin (OVA) can elicit an OVA-specific MHC class I-restricted immune response. J. Immunol., 149:1599 (Sep. 1, 1992). Liposomes were also used by Reddy et. al to incorporate soluble proteins of OVA and .beta.-galactosidase for priming a CD8.sup.+ CTL response to antigen in vivo in mice. J. Immunol., 148:1585 (Mar. 1, 1992), while Bevan et al. have demonstrated CTL priming against soluble OVA with a cell-associated system J. Exp. Med., 171:377 (1990). Complex adjuvants, such as complete Freund's adjuvant (CFA) have also been used with some measure of success. Bacteria, such as Mycobacterium and Staph aureus have also been included as adjuvants in immunization for CTL response. Randall & Young, J. Virol., 65:719-726 (Feb. 1991).
Immune stimulating complexes (ISCOMS), which are multimicellar complexes of cholesterol, phospholipid, and a saponin, have been used fairly extensively as carriers of subunit vaccines, especially in veterinary applications, and have been shown to induce CD8.sup.+ MHC class I-restricted CTL. See, for example, Nadon et al., Vaccine, 10:107 (1992); Mowat et al., J. Immunol. 72:317-322 (1991); Takahashi et al., Nature, 344:873 (Apr. 26, 1990); and Morein, Nature, 322:287 (Mar. 17, 1988). Protective class I MHC-restricted CTLs have been also induced in mice by a recombinant influenza vaccine in an aluminum hydroxide adjuvant, which is currently the only adjuvant licensed by the FDA for clinical use in humans. Dillon et al., Vaccine, 10:309 (1992).
Although the foregoing preparations have been used to induce class I-restricted CTLs with varying degrees of success, none is without potential disadvantages, and those skilled in the area of vaccination biology continue to seek compositions and methods for inducing CTL responses to non-living proteins to confer protective immunity from pathogenic infection and minimize serious side effects. For example, many of the complex adjuvants that have been shown to induce CTLs in laboratory animals are unacceptable for use in domestic animals and humans because of their potential toxicity. Cell-associated formulations do not represent a practical immunization strategy for human vaccines, given the significant possibility for infectious contamination in the preparation. CFA does not routinely achieve priming, even in mice, and may result in tumor formation and tissue necrosis. Antigen encapsulation in liposomes or in a lipid/detergent-based adjuvant, such as the ISCOM matrix, appears to be the most promising approach for immunizing with non-replicating protein, but even this approach may have drawbacks. For example, the ISCOM matrix includes a saponin as an essential component. Saponins are hemolysins and there is an indication in the literature that at least some saponins may be cytotoxic at immunogenic concentrations. In addition, the FDA imposes stringent stability requirements on formulations for human vaccines, and cell associated formulations, liposomes and the ISCOM matrix are potentially problematic from that standpoint.
It is an object of the present invention to provide new compositions and methods for inducing MHC class I-restricted CTLs with non-replicating antigens.